Unless otherwise indicated, all of the saccharides and saccharide residues in this description are the D-form optical isomer, excluding iduronic acid. Also, D-glucosamine (which sometimes includes an N-substituted compound) is sometimes referred to as “GlcN”, and N-acetyl-D-glucosamine is sometimes referred to as “GlcNAc”, D-glucuronic acid is sometimes referred to as “GlcA”, L-iduronic acid is sometimes referred to as “IdoA”, and hexuronic acid representing a uronic acid having 6 carbon atoms, including GlcA and IdoA, is sometimes referred to as “HexA”.
Heparin and heparan sulfate are kinds of glycosaminoglycan having a repeating structure of a disaccharide (4GlcAβ1/IdoAα1→4GlcNAcα1) of HexA residue (GlcA residue or IdoA residue) and GlcNAc residue as the basal skeleton (this basal skeleton may be also referred to as “heparin skeleton” hereinafter), wherein one or more of the 2-position hydroxyl group of its HexA residue and the 2-position amino group, the 3-position hydroxyl group and the 6-position hydroxyl group of its GlcN residue are sulfated.
It has been known so far that the sulfated group of “heparin” or “heparan sulfate” is bound to one or more of the positions shown by R1, R2, R3 and R4 in the following formula (2). However, it has not been known about a glycosaminoglycan in which all of the R1, R2, R3 and R4 are the sulfate group (SO3−) and a production method thereof

On the other hand, it is generally known that a glycosaminoglycan having the heparin skeleton has various physiological activities. For example, it is known for a long time that heparin shows anticoagulation activity upon blood (Thronb. Res., 75, 1–32 (1994)), and it is known also that it has affinity for various growth factors and carries out a role in stabilizing or activating these growth factors (Glycobiology, 4, 451 (1994)). It is known that heparan sulfate also has affinity for various growth factors and accelerates wound healing by stabilizing or activating these growth factors (J. Phthol, 183, 251–252 (1997)). In addition, it is known that a 6-O-desulfated heparin which can be obtained by specifically desulfating only the sulfate group bound to the 6-position of GlcN as a constituting saccharide of heparin is deprived of the anticoagulation activity upon blood but has an action to accelerate wound healing (International Publication WO00/06608), and it is known that a periodic acid oxidation-reduction 2-O-desulfated heparin (mainly keeps the heparin skeleton) which can be obtained by a combination of a periodic acid oxidation-reduction treatment and specific desulfation of the 2-position HexA takes a role in stabilizing various growth factors and accelerating nerve growth (JP-A-11-310602).
Based on these facts, it is considered that the glycosaminoglycan having a heparin skeleton has various physiological activities, and it is considered that derivatives of heparin have markedly large possibilities.
On the other hand, since the gene encoding a glycosaminoglycan sulfotransferase has been cloned, it is considered that information on the substrate specificity of the enzyme for glycosaminoglycan as the sulfate group acceptor can be obtained by preparing the enzyme in a large amount, which will provide a useful approach in studying relationship between the structure and the function of glycosaminoglycan. It is known that there are many sulfation processes in the synthesis of glycosaminoglycan, particularly in the synthesis of heparin/heparan sulfate (Glycotechnology, (5), 57 (1994), published by Kodansha Scientific), and it is considered that various types of glycosaminoglycan sulfotransferases are concerned in this sulfation. Regarding the glycosaminoglycan sulfotransferase which transfers a sulfate group to heparin/heparan sulfate, heparan sulfate N-deacetyl/N-sulfotransferase (hereinafter sometimes referred to as “NDST”), heparan sulfate 2-O-sulfotransferase (hereinafter sometimes referred to as “HS2ST”), heparan sulfate 3-O-sulfotransferase (hereinafter sometimes referred to as “HS3OST”), heparan sulfate 6-O-sulfotransferase (hereinafter sometimes referred to as “HS6ST”) and the like have been isolated from various organisms, particularly from human, and their cDNA molecules have been cloned.
A cDNA of human HS30ST has been disclosed in J. Biol. Chem., 272 28008–28019 (1997), and the cDNA described in the reference has been registered at GenBank as accession number AF019386.
Although an enzyme which can transfer a sulfate group to a glycosaminoglycan having a heparin skeleton is markedly useful because of its high possibility to be used in the enzymatic synthesis of heparin and heparan sulfate, such an enzyme has high substrate specificity so that it is necessary to carry out the synthesis efficiently by using various types of the enzyme for the purpose of industrially synthesizing various types of heparin and heparan sulfate. However, it cannot be said yet that there are sufficient variations of the enzyme capable of transferring a sulfate group to the heparin skeleton.
Accordingly, in the case where production of a glycosaminoglycan having a new structure becomes possible by using an enzyme, it becomes possible to search for a physiological activity possessed by such a glycosaminoglycan.